Proteome profile of human breast cancer tissue generated by LC-ESI-MS/MS combined with sequential protein precipitation and solubilization

Comprehensive proteome profiling of breast cancer tissue samples is challenging, as the tissue samples contain many proteins with varying concentrations and modifications. We report an effective sample preparation strategy combined with liquid chromatography (LC) electrospray ionization (ESI) quadrupole time-of-flight (QTOF) MS/MS for proteome analysis of human breast cancer tissue. The complexity of the breast cancer tissue proteome was reduced by using protein precipitation from a tissue extract, followed by sequential protein solubilization in solvents of different solubilizing strength. The individual fractions of protein mixtures or subproteomes were subjected to trypsin digestion and the resultant peptides were separated by strong cation exchange (SCX) chromatography, followed by reversed-phase capillary LC combined with high resolution and high accuracy ESI-QTOF MS/MS. This approach identified 14407 unique peptides from 3749 different proteins based on peptide matches with scores above the threshold scores at the 95% confidence level in MASCOT database search of the acquired MS/MS spectra. The false positive rate of peptide matches was determined to be 0.95% by using the target-decoy sequence search strategy. On the basis of gene ontology categorization, the identified proteins represented a wide variety of biological functions, cellular processes, and cellular locations.     

Application of Mass Spectrometry in Proteomics

Mass spectrometry has arguably become the core technology in proteomics. The application of mass spectrometry based techniques for the qualitative and quantitative analysis of global proteome samples derived from complex mixtures has had a big impact in the understanding of cellular function. Here, we give a brief introduction to principles of mass spectrometry and instrumentation currently used in proteomics experiments. In addition, recent developments in the application of mass spectrometry in proteomics are summarised. Strategies allowing high-throughput identification of proteins from highly complex mixtures include accurate mass measurement of peptides derived from total proteome digests and multidimensional peptide separations coupled with mass spectrometry. Mass spectrometric analysis of intact proteins permits the characterisation of protein isoforms. Recent developments in stable isotope labelling techniques and chemical tagging allow the mass spectrometry based differential display and quantitation of proteins, and newly established affinity procedures enable the targeted characterisation of post-translationally modified proteins. Finally, advances in mass spectrometric imaging allow the gathering of specific information on the local molecular composition, relative abundance and spatial distribution of peptides and proteins in thin tissue sections.     

Proteomic profiling of hepatocellular carcinoma in Chinese cohort reveals heat-shock proteins (Hsp27, Hsp70, GRP78) up-regulation and their associated prognostic values

To facilitate the identification of candidate molecular biomarkers that are linked to the pathogenesis of hepatocellular carcinoma (HCC), we investigated protein-expression profiles of 146 tissue specimens including 67 pairs of tumors and adjacent non-tumors resected from HCC patients as well as 12 normal livers by 2-DE. Among the 1800 spots displayed in the liver proteome, a total of 90 protein species were found to be significantly different between the three groups (P < 0.05). Three of the top candidate markers up-regulated in HCC, with high receiver operating characteristic (ROC) curves, were identified by MS/MS analysis and belonged to the chaperone members: heat-shock protein (Hsp)27, Hsp70 and glucose-regulated protein (GRP)78. Over-expression of these chaperone proteins in HCC tissues was confirmed by Western blotting and immunohistochemistry. In correlation with clinico-pathological parameters, expression of Hsp27 was linked to alpha-fetoprotein level (P = 0.007) whereas up-regulation of GRP78 was associated with tumor venous infiltration (P = 0.035). No significant association of Hsp70 with any pathologic features was observed. The present HCC proteome analysis revealed that in response to the stressful cancerous microenvironment, tumor cells strived to increase the expression of chaperone proteins for cyto-protective function and to enhance tumor growth and metastasis.     

Proteome profile of bovine ruminal epithelial tissue based on GeLC–MS/MS

The proteome of rumen epithelial tissue was analysed by SDS-PAGE coupled with LC–MS/MS. 813 non-redundant proteins were identified of which 7.4 % featured membrane-spanning domains and 15.4 % harboured a signal peptide. According to the gene ontology annotation, the most abundant proteins exhibited binding activities related to their molecular functions, were proteins of cellular components or belonged to various metabolic processes. A predominant group of canonical pathways in the rumen epithelial tissue was identified using the IPA software. The GeLC–MS/MS approach was used to characterise the entire protein expression repertoire in rumen tissue, providing a more detailed understanding of the important biological processes in the rumen.     

Analysis of the synaptic vesicle proteome using three gel-based protein separation techniques

Synaptic vesicles are key organelles in neurotransmission. Their functions are governed by a unique set of integral and peripherally associated proteins. To obtain a complete protein inventory, we immunoisolated synaptic vesicles from rat brain to high purity and performed a gel-based analysis of the synaptic vesicle proteome. Since the high hydrophobicity of integral membrane proteins hampers their resolution by gel electrophoretic techniques, we applied in parallel three different gel electrophoretic methods for protein separation prior to MS. Synaptic vesicle proteins were subjected to either 1-D SDS-PAGE along with nano-LC ESI-MS/MS or to the 2-D gel electrophoretic techniques benzyldimethyl-n-hexadecylammonium chloride (BAC)/SDS-PAGE, and double SDS (dSDS)-PAGE in combination with MALDI-TOF-MS. We demonstrate that the combination of all three methods provides a comprehensive survey of the proteinaceous inventory of the synaptic vesicle membrane compartment. The identified synaptic vesicle proteins include transporters, soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), synapsins, rab and rab-interacting proteins, additional guanine nucleotide triphosphate (GTP) binding proteins, cytoskeletal proteins, and proteins modulating synaptic vesicle exo- and endocytosis. In addition, we identified novel proteins of unknown function. Our results demonstrate that the parallel application of three different gel-based approaches in combination with mass spectrometry permits a comprehensive analysis of the synaptic vesicle proteome that is considerably more complex than previously anticipated.     

Prefractionation of proteome by liquid isoelectric focusing prior to two-dimensional liquid chromatography mass spectrometric identification

Due to the complexity of proteomes, developing methods of sample fractionation, separation, concentration, and detection have become urgent to the identification of large numbers of proteins, as well as the acquisition of those proteins in low abundance. In this work, liquid isoelectric focusing (LIEF) combined with 2D-LC-MS/MS was applied to the proteome of Saccharomyces cerevisiae. This yielded a total of 1795 proteins that were detected and identified by 30 fractions of protein prefractionation. Categorization of these hits demonstrated the ability of this technology to detect and identify proteins rarely seen in proteome analysis without protein fractionation. LIEF-2D-LC-MS/MS also produced improved resolution of low-abundance proteins. Furthermore, we analyzed the characteristics of proteins obtained by LIEF-2D-LC-MS/MS. 1103 proteins with CAI under 0.2 were identified, allowing us to specifically obtain detailed biochemical information on these kind proteins. It was observed that LIEF-2D-LC-MS/MS is useful for large-scale proteome analysis and may be specifically applied to systems with wide dynamic ranges.     

Simultaneous extraction of nucleic acids and proteins from tissue specimens by ultracentrifugation: A protocol using the high‐salt protein fraction for quantitative proteome analysis

Comprehensive molecular profiling of human tumor tissue specimens at the DNA, mRNA and protein level is often obstructed by a limited amount of available material. Homogenization of frozen tissue samples in guanidine isothiocyanate followed by ultracentrifugation over cesium chloride allows the simultaneous extraction of high‐molecular weight DNA and RNA. Here, we present a protocol for quantitative proteome analysis using the high‐salt protein fraction obtained as supernatant after ultracentrifugation for nucleic acid extraction. We applied this method to extracts from primary human brain tumors and demonstrate its successful application for protein expression profiling in these tumors using 2‐D DIGE, MS and Western blotting.     

Protein biophysical properties that correlate with crystallization success in Thermotoga maritima: maximum clustering strategy for structural genomics

Cost and time reduction are two of the driving forces in the development of new strategies for protein crystallization and subsequent structure determination. Here, we report the analysis of the Thermotoga maritima proteome, in which we compare the proteins that were successfully expressed, purified and crystallized versus the rest of the proteome. This set of almost 500 proteins represents one of the largest, internally consistent, protein expression and crystallization datasets available. The analysis shows that individual parameters, such as isoelectric point, sequence length, average hydropathy, low complexity regions (SEG), and combinations of these biophysical properties for crystallized proteins define a distinct subset of the T. maritima proteome. The distribution profiles of the various biophysical properties in the expression/crystallization set are then used to extract rules to improve target selection and improve the efficiency and output of structural genomics, as well as general structural biology efforts.     

Global organellar proteomics

Cataloging the proteomes of single-celled microorganisms, cells, biological fluids, tissue and whole organisms is being undertaken at a rapid pace as advances are made in protein and peptide separation, detection and identification. For metazoans, subcellular organelles represent attractive targets for global proteome analysis because they represent discrete functional units, their complexity in protein composition is reduced relative to whole cells and, when abundant cytoskeletal proteins are removed, lower abundance proteins specific to the organelle are revealed. Here, we review recent literature on the global analysis of subcellular organelles and briefly discuss how that information is being used to elucidate basic biological processes that range from cellular signaling pathways through protein-protein interactions to differential expression of proteins in response to external stimuli. We assess the relative merits of the different methods used and discuss issues and future directions in the field.     

Exploring the context of the lung proteome within the airway mucosa following allergen challenge

The lung proteome is a dynamic collection of specialized proteins related to pulmonary function. Many cells of different derivations, activation states, and levels of maturity contribute to the changing environment, which produces the lung proteome. Inflammatory cells reacting to environmental challenge, for example from allergens, produce and secrete proteins which have profound effects on both resident and nonresident cells located in airways, alveoli, and the vascular tree which provides blood cells to the parenchyma alveolar bed for gas exchange. In an experimental model of allergic airway inflammation, we have compared control and allergen challenged lung compartments to determine global protein expression patterns using 2D-gel electrophoresis and subsequent spot identification by MS/MS mass spectrometry. We have then specifically isolated the epithelial mucosal layer, which lines conducting airways, from control and allergen challenged lungs, using laser capture technology and performed proteome identification on these selected cell samples. A central component of our investigations has been to contextually relate the histological features of the dynamic pulmonary environment to the changes in protein expression observed following challenge. Our results provide new information of the complexity of the submucosa/epithelium interface and the mechanisms behind the transformation of airway epithelium from normal steady states to functionally activated states.     

Detection and identification of heat shock protein 10 as a biomarker in colorectal cancer by protein profiling

Although colorectal cancer is one of the best-characterized tumors with regard to the multistep progression, it remains one of the most frequent and deadly neoplasms. For a better understanding of the molecular mechanisms behind the process of tumorigenesis and tumor progression, changes in protein expression between microdissected normal and tumorous colonic epithelium were analyzed. Cryostat sections from colorectal tumors, adenoma tissue, and adjacent normal mucosa were laser-microdissected and analyzed using ProteinChip Arrays. The derived MS profiles exhibited numerous statistical differences. One peak showing significantly high expression in the tumor was purified by reverse-phase chromatography and SDS-PAGE. The protein band of interest was passively eluted from the gel and identified as heat shock protein 10 (HSP 10) by tryptic digestion, peptide mapping, and MS/MS analysis. This tumor marker was further characterized by immunohistochemistry. Analysis of HSP 10-positive tissue by ProteinChip technology confirmed the identity of this protein. This work demonstrates that biomarker in colorectal cancer can be detected, identified, and assessed by a proteomic approach comprising tissue microdissection, protein profiling, and immunological techniques. In our experience, histological defined microdissected tissue areas should be used to identify proteins that might be responsible for tumorigenesis.     

Detection of oxidized and glycated proteins in clinical samples using mass spectrometry — A user's perspective

Background

Proteins in human tissues and body fluids continually undergo spontaneous oxidation and glycation reactions forming low levels of oxidation and glycation adduct residues. Proteolysis of oxidised and glycated proteins releases oxidised and glycated amino acids which, if they cannot be repaired, are excreted in urine.

Scope of Review

In this review we give a brief background to the classification, formation and processing of oxidised and glycated proteins in the clinical setting. We then describe the application of stable isotopic dilution analysis liquid chromatography-tandem mass spectrometry (LC-MS/MS) for measurement of oxidative and glycation damage to proteins in clinical studies, sources of error in pre-analytic processing, corroboration with other techniques – including how this may be improved – and a systems approach to protein damage analysis for improved surety of analyte estimations.

Major conclusions

Stable isotopic dilution analysis LC-MS/MS provides a robust reference method for measurement of protein oxidation and glycation adducts. Optimised pre-analytic processing of samples and LC-MS/MS analysis procedures are required to achieve this.

General significance

Quantitative measurement of protein oxidation and glycation adducts provides information on level of exposure to potentially damaging protein modifications, protein inactivation in ageing and disease, metabolic control, protein turnover, renal function and other aspects of body function. Reliable and clinically assessable analysis is required for translation of measurement to clinical diagnostic use. Stable isotopic dilution analysis LC-MS/MS provides a “gold standard” approach and reference methodology to which other higher throughput methods such as immunoassay and indirect methods are preferably corroborated by researchers and those commercialising diagnostic kits and reagents. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.     

BiomarkerDigger: A versatile disease proteome database and analysis platform for the identification of plasma cancer biomarkers

We have developed a proteome database (DB), BiomarkerDigger ( http://biomarkerdigger.org ) that automates data analysis, searching, and metadata‐gathering function. The metadata‐gathering function searches proteome DBs for protein–protein interaction, Gene Ontology, protein domain, Online Mendelian Inheritance in Man, and tissue expression profile information and integrates it into protein data sets that are accessed through a search function in BiomarkerDigger. This DB also facilitates cross‐proteome comparisons by classifying proteins based on their annotation. BiomarkerDigger highlights relationships between a given protein in a proteomic data set and any known biomarkers or biomarker candidates. The newly developed BiomarkerDigger system is useful for multi‐level synthesis, comparison, and analyses of data sets obtained from currently available web sources. We demonstrate the application of this resource to the identification of a serological biomarker for hepatocellular carcinoma by comparison of plasma and tissue proteomic data sets from healthy volunteers and cancer patients.     

Evaluation of three principally different intact protein prefractionation methods for plasma biomarker discovery

The aim of this study was to evaluate three principally different top-down protein prefractionation methods for plasma: high-abundance protein depletion, size fractionation and peptide ligand affinity beads, focusing in particular on compatibility with downstream analysis, reproducibility and analytical depth. Our data clearly demonstrates the benefit of high-abundance protein depletion. However, MS/MS analysis of the proteins eluted from the high-abundance protein depletion column show that more proteins than aimed for are removed and, in addition, that the depletion efficacy varies between the different high-abundance proteins. Although a smaller number of proteins were identified per fraction using the peptide ligand affinity beads, this technique showed to be both robust and versatile. Size fractionation, as performed in this study, focusing on the low molecular weight proteome using a combination of gel filtration chromatography and molecular weight cutoff filters, showed limitations in the molecular weight cutoff precision leading detection of high molecular weight proteins and, in the case of the cutoff filters, high variability. GeLC-MS/MS analysis of the fractionation methods in combination with pathway analysis demonstrates that increased fractionation primarily leads to high proteome coverage of pathways related to biological functions of plasma, such as acute phase reaction, complement cascade and coagulation. Further, the prefractionation methods in this study induces limited effect on the proportion of tissue proteins detected, thereby highlighting the importance of extensive or targeted downstream fractionation.     

Proteomic Profiling and Neurodegeneration in Alzheimer's Disease

Quantitative proteome analysis of Alzheimer's disease (AD) brains was performed using 2-D gels to identify disease specific changes in protein expression. The task of characterizing the proteome and its components is now practically achievable because of the development and integration of four important tools: protein, EST, and complete genome sequence databases, mass spectrometry, matching software for protein sequences and protein separation technology. Mass spectrometry (MS) instrumentation has undergone a tremendous change over the past decade, culminating in the development of highly sensitive, robust instruments that can reliably analyze biomolecules, particularly proteins and peptides; we identified 35 proteins from over 100 protein spots on a 2-D gel. Using this current technology, protein-expression profiling, which is actually a specialized form of mining, is an important principal application of proteomics. The information obtained has tremendous potential as a means of determining the pathogenesis, and detecting disease markers and potential targets for drug therapy in AD.